Shuttle vectors

ABSTRACT

The invention provides shuttle vectors, and methods of using shuttle vectors, capable of expression in, at least, a mammalian cell. Furthermore, the shuttle vectors are capable of replication in at least yeast, and optionally, bacterial cells. Also provided is a method wherein yeast are transformed with a shuttle vector as provided herein. Heterologous nucleic acids flanked by 5′ and 3′ ends identical to a homologous recombination site within the shuttle vector are introduced to the transformed yeast and allowed to homologously recombine with the shuttle vector such that they are inserted into the vector by the yeast organism. The shuttle vector is then recovered and transferred to a mammalian cell for expression.

This is a continuation-in-part of application Ser. No. 09/133,944 filed Aug. 14, 1998, now U.S. Pat. No. 6,280,937.

FIELD OF THE INVENTION

The invention relates to novel shuttle vectors, and more particularly, shuttle vectors capable of replication in at least yeast and capable of expression in at least a mammalian cell.

BACKGROUND OF THE INVENTION

The introduction of cloned nucleotide sequences into mammalian cells has greatly facilitated the study of the control and function of various eukaryotic genes. Mammalian cells provide an environment conducive to appropriate protein folding, post translational processing, feedback control, protein-protein interactions, and other eukaryotic protein modifications such as glycosylation and oligomerization. Thus, a number of expression vectors have been developed which allow the expression of a polypeptide in a mammalian cell.

The typical mammalian expression vector will contain (1) regulatory elements, usually in the form of a viral promoter or enhancer sequences; (2) a multicloning site, usually having specific enzyme restriction sites to facilitate the insertion of a DNA fragment with the vector; and (3) sequences responsible for intron splicing and polyadenylation of mRNA transcripts. Generally, sequences facilitating the replication of the vector in both bacterial and mammalian hosts and a selection marker gene which allows selection of transformants in bacteria are also included. The bacterial elements, or in some cases phage elements, are included to provide the option of further analyzation of the nucleic acid inserts amplified and isolated from the bacteria or phage.

In the past, the insertion of a heterologous nucleic acid (insert) into the multicloning site of a mammalian expression vector has generally been accomplished by one of two methods. In the first method, the insert is cut out of a bacterial expression vector and ligated into the mammalian expression vector. In the second method, often called “TA cloning”, special ends are generated on the insert by PCR such that the modified insert can be put into the mammalian expression vector. Each of these methods requires a number of steps including enzymatic reactions which can be labor intensive and unreliable. Moreover, cloning efficiency drops significantly as the size of the insert increases.

Another method used for inserting a heterologous nucleic acid (insert) into an expression vector takes advantage of yeast's high efficiency at homologous recombination in vivo. In this method, a nucleic acid fragment flanked by 5′ and 3′ homologous regions is co-introduced into a yeast with a vector which has regions identical to the 5′ and 3′ regions flanking the fragment. The yeast efficiently homologously recombines such that the fragment inserts into the region of the vector flanked by the before-mentioned 5′ and 3′ regions. H.a., et al., Plasmid, 38:91-96 (1997), incorporated herein. Unfortunately, yeast are the only organisms able to efficiently recombine so as to insert heterologous nucleic acids into a vector. Therefore, to date, there is not an efficient method or means to transfer inserts into a specific region of a vector used for expression in mammalian cells.

Accordingly, it is an object of the invention to provide compositions and methods useful in facilitating the insertion of a heterologous nucleic acid into a vector which can express the heterologous nucleic acid in at least a mammalian cell.

Moreover, it is the object of this invention to provide a shuttle vector and methods of use which allow replication of the shuttle vector at least in yeast and which allow expression in at least a mammalian cell.

SUMMARY OF THE INVENTION

The invention provides shuttle vectors, and methods of using shuttle vectors, capable of expression in at least a mammalian cell. Furthermore, the shuttle vectors are capable of replication in at least yeast, and optionally, bacterial cells.

In one aspect of the invention, the invention provides a shuttle vector comprising an origin of replication functional in yeast and preferably, a reporter gene functional in yeast. The shuttle vector further comprises a promoter functional in a mammalian cell, capable of directing transcription of a polypeptidc coding sequence operably linked to said promoter.

In another aspect of the invention, the shuttle vector comprises an insertion site operably linked to said promoter. The insertion site preferably allows for homologous recombination with a heterologous nucleic acid. In one embodiment, the insertion site has 5′ and 3′ regions identical to 5′ and 3′ regions flanking a nucleic acid to be inserted into the vector.

Optionally, the shuttle vector comprises any one or more of the following: an internal ribosome entry sequence (IRES), a polyadenylation sequence and a splice sequence.

In another aspect of the invention, the shuttle vector further comprises an origin of replication functional in a bacterial cell and preferably, a selectable gene functional in a bacterial cell. The shuttle vector may also comprise an origin of replication functional in a mammalian cell, and optionally, a selectable gene functional in a mammalian cell.

The present invention also provides methods for using the shuttle vectors provided herein. In one embodiment, heterologous nucleic acids flanked by regions identical to flanking regions of the insertion site within a shuttle vector are co-introduced to yeast with the shuttle vector and allowed to homologously recombine such that the heterologous nucleic acids are inserted into the shuttle vector by the yeast organism. In preferred embodiments, the heterologous nucleic acids are introduced to the yeast in a linear nucleic acid. The shuttle vector is then recovered and transferred to a mammalian cell for expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a shuttle vector in accordance with the present invention referred to herein as pPYC-R. The following abbreviations are used herein: IRES for internal ribosomal binding site, GFP for a green florescence protein, Amp^(R) for an Ampicillin resistance gene, TRP for a tryptophan gene, and MCS for multi-cloning site or sequence.

FIGS. 2A through 2D show the nucleotide sequence (SEQ ID NO:1) of pPYC-R. In SEQ ID NO:1, a CMV promoter is at nucleotides 4853-5614, an IRES is at nucleotides 6001-6505, a GFP gene is at nucleotides 6506-7258, an Amp^(R) gene is at nucleotides 9888-655, an E. coli origin of replication site is at nucleotides 656-1456, a yeast 2 μ origin of replication is at nucleotides 1461-2808, and a TRP gene is at nucleotides 3344-4018. The intron contains 5′ and 3′ splice sites.

FIG. 3 is a photograph showing the results of a mammalian cell transfection assay using the shuttle vector pPYC-R.

FIG. 4 is a schematic of a shuttle vector in accordance with the present invention referred to herein as pPYC.

FIGS. 5A through 5D show the nucleotide sequence (SEQ ID NO:2) of pPYC. In SEQ ID NO:2, a CMV promoter is at nucleotides 1-750, an IRES site is at nucleotides 1158-1662, a GFP gene is at nucleotides 1683-2402, a yeast 2μ origin of replication is at nucleotides 2985-4332, a tryptophan gene is at nucleotides 4868-5542, an Ampicillin resistance gene is at nucleotides 5982-6842, and an E. coli origin or replication is at nucleotides 7142-7669. The tag is hemagglutinin (HA).

FIG. 6 is a photograph showing pPYC transfection wherein pPYC includes a gene causing apoptosis in accordance with the present invention. The photograph shows extensive cell death due to the expression of HA tagged Rip.

FIG. 7 is a schematic of a shuttle vector in accordance with the present invention referred to herein as pCRU5YMS.

FIGS. 8A through 8C show the nucleotide sequence (SEQ ID NO:5) of pCRU5YMS. At nucleotides 1-664 is the CMV promoter; at nucleotides 2197-2725 is the IRES. At nucleotides 2746-3465 is the GFP gene; at nucleotides 3522-4252 is the LTR; and at nucleotides 4253-5500 is the Yeast selection marker TRP gene. At nucleotides 5512-6860 is the Yeast 2 micron replication origin; at nucleotides 6861-7650 is the E. coli replication origin and at nucleotides 7678-8538 is the Ampicillin resistance gene.

FIG. 9 is a photograph showing the results of a mammalian cell transfection assay using the shuttle vector pCRU5YMS.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides shuttle vectors, and methods of use, wherein the shuttle vectors are capable of expression in at least a mammalian cell and capable of replication in at least yeast. In the past, vectors have been constructed so as to be functional in certain aspects either in mammalian cells or yeast, but not both. As described herein, different hosts provide different advantages to an expression vector and in particular, to the expression product. By providing a vector which is functional as described herein in multiple hosts, the invention allows the advantages provided by varying hosts to be obtained by the use of a single tool.

For example, a vector having the ability to replicate in yeast is useful for a variety of reasons. An advantage of the yeast system is its efficiency at homologous recombination. Orr-Weaver, et al., PNAS USA, 80:4417-4421 (1983), incorporated herein by reference. By taking advantage of yeast's ability to insert heterologous nucleic acids into a vector, this eliminates the steps of manipulating the ends of the vector and the heterologous nucleic acid and ligating the two together. Another advantage of this system is that yeast can be transformed with large nucleic acids, i.e., up to at least 10 kilobases, which can then be inserted into the vector.

Moreover, yeast is a well-studied organism which facilitates its use. In particular, yeast has been widely used to detect protein-protein interactions in the “two-hybrid system”. The two-hybrid system is a method used to identify and clone genes for proteins that interact with a protein of interest. Briefly, the system indicates protein-protein interaction by the reconstitution of GAL4 function, which is detectable and only occurs when the proteins interact. This system and general methodologies concerning the transformation of yeast with expressible vectors are described in Cheng-Ting et al., PNAS USA, 88:9578-9582 (1991), Fields and Song, Nature, 340:245-246 (1989), and Chevray and Nathans, PNAS USA, 89:5789-5793 (1992), each incorporated herein in their entirety.

Regarding mammalian cells, these cells are preferred for the expression of eukaryotic proteins particularly when determining or studying the function of the protein. Mammalian cells are able to reproduce the protein's proper glycosylation and oligomerization, folding, post translational processing, feedback control, protein-protein interaction, etc., and thus are advantageous for expression of eukaryotic and particularly mammalian proteins.

Regarding bacterial cells or phage, these systems are also very well studied and are therefore easily manipulated. In particular, bacteria and phage are useful for the rapid amplification of nucleic acids.

Thus, while vectors which function in one of yeast, mammalian cells, bacteria or phage are useful, vectors which can successfully shuttle between these systems are particularly desirable. This invention provides such vectors. In a preferred embodiment, the shuttle vector functions in both yeast and mammalian cells. Such a shuttle vector can allow for the exploitation of the yeast two-hybrid system as well as yeast's ability to homologously recombine, as well as provide a convenient means for subsequent expression in mammalian cells to, for example, verify protein-protein interactions, study the protein's function, etc.

In one embodiment, the invention provides a shuttle vector comprising an origin of replication functional in yeast and a promoter functional in a mammalian cell. Preferably, the shuttle vector also comprises a selectable gene functional in yeast.

The origin of replication functional in yeast is any nucleic acid sequence which allows replication of the shuttle vector independently from the chromosome. Generally, the origin of replication is functional in at least one or more of the following: Saccharomyces cerevisiae, Candida albicans and C. iialtosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia gitillerirnondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Suitable origin of replication sites include, for example, ars 1, centromere ori, and 2 μ ori. Yeast origin of replication sites can be used to increase the copy number and to retrieve the vector from yeast.

The “promoter functional in a mammalian cell” or “mammalian promoter” is capable of directing transcription of a polypeptide coding sequence operably linked to said promoter. The choice of the promoter will depend in part on the mammalian cell into which the vector is put. Generally, this promoter is functional in at least one or more of the following: Chinese hamster ovary (CHO), BHK, 293, Hela, NH3T3 and COS cells. More specific examples include monkey kidney CV1 line; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriate host cell is deemed to be within the skill in the art.

Transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as adenoviruses, retroviruses, lentiviruses, herpes viruses, including but not limited to, polyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989), adenovirus 2, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), hepatitis-B virus, Simian Virus 40 (SV40), Epstein Barr virus (EBV), feline immunedcficicncy virus (FIV), and Srα, or are respiratory synsitial viral promoters (RSV) or long terminal repeats (LTRs) of a retrovirus, i.e., a Moloney Murine Leukemia Virus (MoMuLv) (Cepko et al. (1984) Cell 37:1053-1062). Moreover, the promoters can be selected from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, and functional derivatives thereof, provided such promoters are compatible with the host cell systems. The promoter functional in a mammalian cell can be inducible or constitutive.

In an embodiment provided herein, the shuttle vector is double stranded and on a first strand, comprises a first promoter operably linked to either a coding sequence or a site for the insertion of a coding sequence of interest (i.e., a heterologous nucleic acid) followed by a polyadenylation site. On a second strand, the shuttle vector comprises two LTRs flanking said region comprising said first promoter and coding sequence or cloning site, wherein the LTRs operate in a direction opposite to said first promoter.

“Operably linked” as used herein means that the transcriptional and translational regulatory nucleic acid is positioned relative to any coding sequences in such a manner that transcription is initiated. Generally, this will mean that the promoter and transcriptional initiation or start sequences are positioned 5′ to the coding region. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used, as will be appreciated by those in the art.

By “vector” or “episome” herein is meant a nucleic acid replicon used for the transformation of host cells. The vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a mammalian host genome, such as a retroviral based vector. In a preferred embodiment, the shuttle vector remains as an extrachromosomal vector in bacteria and yeast, and is integrated into the genome of the mammalian cell.

A preferred embodiment utilizes retroviral desired vectors. Currently, the most efficient gene transfer methodologies harness the capacity of engineered viruses, such as retroviruses, to bypass natural cellular barriers to exogenous nucleic acid uptake. The use of recombinant retroviruses was pioneered by Richard Mulligan and David Baltimore with the Psi-2 lines and analogous retrovirus packaging systems, based on NIH 3T3 cells (see Mann et al., Cell 33:153-159 (1993), hereby incorporated by reference). Such helper-defective packaging lines are capable of producing all the necessary trans proteins—gag, pol, and env—that are required for packaging, processing, reverse transcription, and integration of recombinant genomes. Those RNA molecules that have in cis the ψ packaging signal are packaged into maturing virions. In addition, transfection efficiencies of retroviruses can be extremely high, thus obviating the need for selection genes in some cases.

Retroviral transfection systems are further described in Mann et al., supra: Pear et al., PNAS USA 90(18):8392-6 (1993); Kitamura et al., PNAS USA 92:9146-9150 (1995); Kinsella et al., Human Gene Therapy 7:1405-1413; Hoffmann et al., PNAS USA 93:5185-5190; Choate et al., Human Gene Therapy 7:2247 (1996); WO 94/19478; PCT US97/01019, and references cited therein, all of which are incorporated by reference.

Any number of suitable retroviral vectors may be used to construct the shuttle vectors of the invention. Preferred retroviral vectors include a vector based on the murine stem cell virus (MSCV) (see Hawley et al., Gene Therapy 1 :136 (1994)) and a modified MFG virus (Rivere et al., Genetics 92:6733 (1995)), and pBABE (see PCT US97/01019, incorporated by reference), and functional derivatives thereof.

In addition, it is possible to configure a retroviral vector to allow expression of genes after integration in target cells. For example, Tet-inducible retroviruses can be used to express genes (Hoffman et al., PNAS USA 93:5185 (1996)). Expression of this vector in cells is virtually undetectable in the presence of tetracycline or other active analogs. However, in the absence of Tet, expression is turned on to maximum within 48 hours after induction, with uniform increased expression of the whole population of cells that harbor the inducible retrovirus, indicating that expression is regulated uniformly within the infected cell population.

The shuttle vector can also be based on a non-retroviral vector. Any number of known vectors are suitable, including, but not limited to, pREP9, pCDNA, pCEP4 (Invitrogen), pCI and pCI-NEO (Promega). Basically, any vector can be reconstructed to contain the components as described herein. For example, construction of suitable vectors containing the components described herein can be achieved by employing standard ligation techniques which are known to the skilled artisan, using cloned or synthetic sequences.

In a preferred embodiment, the shuttle vector includes a selectable gene functional in yeast (also referred to herein as a yeast reporter gene). By “selectable gene” or “reporter gene” herein is meant a gene that by its presence in a host cell, i.e. upon expression, can allow the host to be distinguished from a cell that does not contain the selectable gene. Selectable genes can be classified into several different types, including survival and detection genes. It may be the nucleic acid or the protein expression product that causes the effect. Additional components, such as substrates, ligands, etc., may be additionally added to allow selection or sorting on the basis of the selectable gene.

In a preferred embodiment, the selectable gene is a survival gene that serves to provide a nucleic acid (or encode a protein) without which the cell cannot survive, such as a drug resistant gene, a growth regulatory gene, or a nutritional requirement. The selectable gene functional in yeast is preferably a survival gene. Wherein a selectable gene functional in bacteria is included in the shuttle vector, a survival gene is also preferred.

Preferred survival genes functional in yeast are survival genes which include ADE2, HIS3, LEU2, TRP 1, URA3, and ALG7, which confer resistance to tunicamycin; the neomycin phosphotransferase gene, which confers resistance to G418; the CUP1 gene, which allows yeast to grow in the presence of copper ions; and an adenine producing gene, or the like, which may be used alone or in combinations of two or more thereof. In a preferred embodiment, the trp1 gene is utilized. Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10: 157 (1980). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. The preferred selectable gene functional in bacteria is a drug resistant, such as an ampicillin resistant gene.

In a preferred embodiment, the selectable gene is a detection gene. Wherein a selectable gene functional in mammalian cells is included in the vector, a detection gene is preferred. Detection genes encode a protein that can be used as a direct or indirect label, i.e., for sorting the cells, i.e. for cell enrichment by FACS. In this embodiment, the protein product of the selectable gene itself can serve to distinguish cells that are expressing the selectable gene. In this embodiment, suitable selectable genes include those encoding green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), luciferase, β-galactosidase, all commercially available, i.e., Clontech, Inc.

Alternatively, the selectable gene encodes a protein that will bind a label that can be used as the basis of selection; i.e. the selectable gene serves as an indirect label or detection gene. In this embodiment, the selectable gene should encode a cell-surface protein. For example, the selectable gene may be any cell-surface protein not normally expressed on the surface of the cell, such that secondary binding agents could serve to distinguish cells that contain the selectable gene from those that do not. Alternatively, albeit non-preferably, selectables comprising normally expressed cell-surface proteins could be used, and differences between cells containing the selectable construct and those without could be determined. Thus, secondary binding agents bind to the selectable protein. These secondary binding agents are preferably labeled, for example with fluors, and can be antibodies, haptens, etc. For example, fluorescently labeled antibodies to the selectable gene can be used as the label. Similarly, membrane-tethered streptavidin could serve as a selectable gene, and fluorescent biotin could be used as the label, i.e. the secondary binding agent. Alternatively, the secondary binding agents need not be labeled as long as the secondary binding agent can be used to distinguish the cells containing the construct; for example, the secondary binding agents may be used in a column, and the cells passed through, such that the expression of the selectable gene results in the cell being bound to the column, and a lack of the selectable gene (i.e. inhibition), results in the cells not being retained on the column. Other suitable selectable proteins/secondary labels include, but are not limited to, antigens and antibodies, enzymes and substrates (or inhibitors), etc.

In one aspect of the invention, the shuttle vector includes an insertion site, which is used to insert a heterologous nucleic acid sequence of choice, for ultimate expression in mammalian cells. The insertion site can be either be a cloning site, preferably a multicloning site (MCS), or a site suitable for homologous recombination, (referred to herein as a homologous recombination site). The vector can include multiple insertion sites, including both cloning sites and at least one homologous recombination site.

In a preferred embodiment, the insertion site is a cloning site. A cloning site as used herein is a known sequence, preferably the only one on the vector, (i.e., it is a unique sequence on the vector) upon which a restriction enzyme operates to linearize or cut the vector. A multicloning site, also sometimes referred to as a multiple cloning site, polylinker, or polycloning site, is a cluster of cloning sites such that many restriction enzymes operate thereon. A wide variety of these sites are known in the art.

In a preferred embodiment, the insertion site is a site that allows the introduction of the heterologous nucleic acid into the shuttle vector by homologous recombination. Homologous recombination is, briefly, the process of strand exchange that can occur spontaneously with the alignment of homologous sequences (i.e. sets of complementary strands). As is known in the art, yeast are efficient at homologous recombination. Orr-Weaver, et al, supra; H.a., et al., supra; Ma, et al., Gene, 58:201-216 (1987); Petermann, Nucleic Acids Res., 26(9):2252-2253 (1998); each incorporated herein by reference. Thus, in general, the homologous recombination site contains two distinct, but generally contiguous, regions. The first region, referred to herein as the 5′ region, is generally identical to the 5′ region flanking the heterologous nucleic acid to be inserted into the vector. The second region, referred to herein as the 3′ region, is generally identical to the 3′ region flanking the heterologous nucleic acid to be inserted into the vector. Preferably, the 5′ and 3′ regions are each at least 12 or 15 nucleic acids long. More preferably, the 5′ and 3′ regions are each at least about 20 or 30 nucleic acids long, and more preferably at least about 50 nucleic acids long, and most preferably about 60 nucleic acids long. These regions are preferably less than about 100 nucleic acids long. Preferably, the homologous recombination site sequence is unique to the vector in that the vector does not comprise another sequence corresponding to the sequence of the homologous recombination site.

The insertion site is used to insert a heterologous nucleic acid. A “heterologous nucleic acid” as used herein refers to any nucleic acid inserted into the shuttle vector at a site operably linked to the promoter. Various embodiments of heterologous nucleic acids are further defined below. In a preferred embodiment, the heterologous nucleic acid is flanked by 5′ and 3′ regions identical to the 5′ and 3′ regions of a homologous recombination site on the shuttle vector provided herein. Thus, when the heterologous nucleic acid is inserted into the vector, the 5′ and 3′ regions flanking the heterologous nucleic acid replace the 5′ and 3′ regions of the homologous recombination site during homologous recombination.

In a further aspect, the shuttle vector further comprises an origin of replication functional in a bacterial cell. The bacterial cell is generally any bacterial cell which can be used to amplify the shuttle vector. Examples include Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli, Bacillus subtilis, Streptococcus cremoris, Streptococcus lividans. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Origin of replication sites are known in the art and are further described in Sambrook, et al., MolecularCloting, 2nd Ed., Vol. 3, Chapter 1, particularly sections 12-20 (1989), Promega, 1998 catalog number E1841 (pCI-neo).

In one embodiment, the shuttle vector also comprises an origin of replication functional in mammalian cells. As is known in the art, the only extrachromosomal vectors which replicate in mammalian cells are virally derived. A number of viral origin of replications require the binding of a specific viral replication protein to effect replication. Suitable origin of replication/viral replication protein pairs include, but are not limited to, the Epstein Barr origin of replication and the Epstein Barr nuclear antigen (see Sugden et al., Mole. Cell. Biol. 5(2):410-413 (1985)); the SV40 origin of replication and the SV40 T antigen (see Margolskee et al., Mole. Cell. Biol. 8(7):2837 (1988)). The coding sequence for the viral replication protein can be on the shuttle vector provided herein, or on a separate vector.

In an additional aspect of this invention, the shuttle vector comprises additional sequences, including but not limited to at least one or all of the following: an internal ribosome entry sequence (IRES), an RNA splice site (also called a splice signal or sequence herein) and a polyadenylation site (also called a polyadenylation signal or sequence herein).

IRES elements function as initiators of the efficient translation of reading frames. In particular, IRES allows for the translation of two different genes on a single transcript. IRES thus greatly facilitates the selection of cells expressing peptides at uniformly high levels. IRES elements are known in the art and are further characterized in Kim, et al., Molecular and Cellular Biology 12(8):3636-3643 (August 1992) and McBratney, et al., Current Opinion in Cell Biology 5:961-965 (1993).

All of those sequences of viral, cellular, or synthetic origin which mediate an internal binding of the ribosomes can be used as an IRES. Examples include those IRES elements from poliovirus Type I, the 5′UTR of encephalomyocarditis virus (EMV), of “Thelier's murine encephalomyelitis virus (TMEV) of “foot and mouth disease virus” (FMDV) of “bovine enterovirus (BEV), of “coxsackie B virus” (CBV), or of “human rhinovirus” (HRV), or the “human immunoglobulin heavy chain binding protein” (BIP) 5′UTR, the Drosophila antennapediae 5′UTR or the Drosophila ultrabithorax 5′UTR, or genetic hybrids or fragments from the above-listed sequences.

The shuttle vectors provided herein may include a splice donor and acceptor site (splicing signals or splice sites) within the transcription unit. Splicing signals are known to increase mRNA stability and protein expression levels. Splicing signals are known in the art and are further described in Sambrook, et al., Molecular Cloning, 2nd Ed., Vol. 3, Chapter 16, particularly section 7 (1989).

A polyadenylation site or signal refers to sequences necessary for the termination of transcription and for stabilizing the mRNA of eukaryotes. Such sequences are commonly available and are further described in Sambrook, et al., Molecular Cloning, 2nd Ed., Vol. 3, Chapter 16, particularly sections 6-7 (1989).

Optionally, the shuttle vector may further comprise transcription enhancers. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes including for example, globin, elastase, albumin, α-fetoprotein, and insulin. Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5′ or 3′ to a coding sequence, but is preferably located at a site 5′ from the promoter.

Optionally, the vector can be constructed so as to allow of the heterologous nucleic acid expression in yeast and/or bacterial cells. In this embodiment, the vector would further include a promoter functional in yeast and/or bacterial cells. Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.

Promoters for bacterial cells are known in the art and further described i.e., in Sambrook, et al., Molecular Cloning, 2nd Ed., Vol. 3, Chapter 17, particularly sections 11-17 (1989). Generally, promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence.

In an embodiment provided herein, the insertion site is linked to a selection system, i.e., a detection gene. In a preferred embodiment, from the 5′ to 3′ direction the construct comprises the mammalian promoter, the heterologous nucleic acid, the IRES site, and the selectable gene.

In a preferred embodiment, the vectors are used to screen heterologous nucleic acids. “Heterologous nucleic acids” as used herein refers to naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids, e.g. in nucleotide/residue frequency generally or per position. By “randomized” or grammatical equivalents herein is meant that each nucleic acid consists of essentially random nucleotides. For example, digests of procaryotic or eukaryotic genomes may be used, or cDNA fragments. They are heterologous in that they are inserted into the shuttle vector.

In a preferred embodiment, the heterologous nucleic acids are presented to the shuttle vector in the form of a cloning vector wherein the heterologous nucleic acid is flanked by 5′ and 3′ regions identical to 5′ and 3′ regions of an insertion site (i.e., a homologous recombination site) on the shuttle vector. That is, heterologous nucleic acids are recombined into cloning vectors containing homologous recombination flanking regions. The cloning vectors and the shuttle vectors are introduced into yeast, where recombination takes place. In a preferred embodiment, the cloning vectors are linear when introduced to the yeast.

In one aspect of the invention, the shuttle vectors provided herein are used to transform yeast. Heterologous nucleic acids are then, or simultaneously introduced to the yeast, and in a preferred embodiment, homologous recombination takes place such that the yeast inserts the heterologous nucleic acid into the shuttle vector at a specific insertion site, i.e., a homologous recombination site.

Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). The shuttle vectors are then isolated from the yeast and used to transform mammalian cells for expression of the heterologous nucleic acid.

For transforming mammalian cells, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transformations have been described in U.S. Pat. No. 4,399,216. However, other methods for introducing DNA into cells, such as by nuclear microinjection, biolistics, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988). Expression in mammalian cells is also described in Sambrook, et al., Molecular Cloning, 2nd Ed., Vol. 3, Chapter 16, particularly sections 68-72 (1989).

Isolation of the shuttle vectors is performed by standard techniques known in the art. Generally, the shuttle vectors can be isolated by breaking the cell open and separating the vector nucleic acid based on weight, i.e., centrifugation, or size, i.e. gel permeability. The vectors need only be isolated to the extent required to perform transformation.

In one aspect of this invention, the invention involves expression of heterologous nucleic acid inserts in a mammalian cell population. The expression of heterologous nucleic acids is identified by the production of a label or tag. Thus, when the shuttle vector expresses a heterologous nucleic acid, a selectable gene will also be expressed thereby verifying the presence of an expressed heterologous nucleic acid.

In another aspect of the present invention, expressed heterologous nucleic acids are selected on the basis of activity or phenotype. For example, the expressed insert or the cell type expressing that particular insert can be screened for its ability to interact with an antibody or ligand, capable of specific binding to the encoded product of that insert, which has been previously bound to a solid support such as a petri dish. Positive CDNA inserts (those expressed in cell types binding to the solid support) are recovered, transformed into a convenient host (E. coli) and characterized by known recombinant DNA techniques. This procedure is also referred to as panning, and is further described in Wysocki and Sata, 1979 PNAS 75:2844-2848 and Seen and Aruffo, 1987 PNAS 84:3365-3369.

Thus, in one embodiment, the present invention allows for creating shuttle vectors with inserts therein, without necessarily requiring the skilled artisan to insert the heterologous nucleic acid into the shuttle vector. Rather, the invention herein provides for the yeast organism to perform this step in a preferred embodiment. Moreover, the present invention also allows for expression in mammalian cells, which provides for a native environment for expressing mammalian genes. Additionally, the invention provides for a variety of options, such as replication in bacteria for amplification of shuttle vectors containing selected heterologous nucleic acids. Moreover, the shuttle vectors provided herein can perform the traditional aspects of expression vectors, whether or not “shuttling” is desired.

Furthermore, the present invention provides for screening for heterologous nucleic acids which encode candidate agents. “Candidate agents” as used herein are peptides which may have a desired effect on the phenotype or genotype of a cell. Heterologous nucleic acids expressing a candidate agent can be designed in a number of ways so as to facilitate their identification. Generally, this is achieved by the use of fusion partners, or combinations of fusion partners. Examples include presentation structures, targeting sequences, rescue sequences, and stability sequences, all of which can be used independently or in combination, with or without linker sequences.

By “fusion partner” or “functional group” herein is meant a sequence that is associated with the heterologous nucleic acid expressing a candidate agent, that confers upon all members of the library in that class a common function or ability. Fusion partners can be heterologous (i.e. not native to the host cell), or synthetic (not native to any cell). Suitable fusion partners include, but are not limited to: a) presentation structures, as defined below, which provide the candidate bioactive agents in a conformationally restricted or stable form; b) targeting sequences, defined below, which allow the localization of the candidate bioactive agent into a subcellular or extracellular compartment; c) rescue sequences as defined below, which allow the purification or isolation of either the candidate bioactive agents or the nucleic acids encoding them; d) stability sequences, which confer stability or protection from degradation to the candidate bioactive agent or the nucleic acid encoding it, for example resistance to proteolytic degradation; e) dimerization sequences, to allow for peptide dimerization; or f) any combination of a), b), c), d), and e), as well as linker sequences as needed.

In a preferred embodiment, the fusion partner is a presentation structure. By “presentation structure” or grammatical equivalents herein is meant a sequence, which, when fused to a heterologous nucleic acid expressing a candidate agent, causes the candidate agents to assume a conformationally restricted form. Proteins interact with each other largely through conformationally constrained domains. Although small peptides with freely rotating amino and carboxyl termini can have potent functions as is known in the art, the conversion of such peptide structures into pharmacologic agents is difficult due to the inability to predict side-chain positions for peptidomimetic synthesis. Therefore the presentation of peptides in conformationally constrained structures will benefit both the later generation of pharmaceuticals and will also likely lead to higher affinity interactions of the peptide with the target protein. This fact has been recognized in the combinatorial library generation systems using biologically generated short peptides in bacterial phage systems. A number of workers have constructed small domain molecules in which one might present randomized peptide structures.

Suitable presentation structures include, but are not limited to, minibody structures, loops on beta-sheet turns and coiled-coil stem structures in which residues not critical to structure are randomized, zinc-finger domains, cysteine-linked (disulfide) structures, transglutaminase linked structures, cyclic peptides, B-loop structures, helical barrels or bundles, leucine zipper motifs, etc.

In a preferred embodiment, the presentation structure is a coiled-coil structure, allowing the presentation of the randomized peptide on an exterior loop. See, for example, Myszka et al., Biochem. 33:2362-2373 (1994), hereby incorporated by reference). Using this system investigators have isolated peptides capable of high affinity interaction with the appropriate target. In general, coiled-coil structures allow for between 6 to 20 randomized positions.

A preferred coiled-coil presentation structure is as follows:

MGCAALESEVSALESEVALESEVAALGRGDMPLAAVKSKLSAVKSKLAS VKSKLAACGPP (SEQ ID NO. 6. The underlined regions represent a coiled-coil leucine zipper region defined previously (see Martin et al., EMBO J. 13(22):5303-5309 (1994), incorporated by reference). The bolded GRGDMP region represents the loop structure and when appropriately replaced with randomized peptides (i.e.candidate bioactive agents, generally depicted herein as (X)_(n), where X is an amino acid residue and n is an integer of at least 5 or 6) can be of variable length. The replacement of the bolded region is facilitated by encoding restriction endonuclease sites in the underlined regions, which allows the direct incorporation of randomized oligonucleotides at these positions. For example, a preferred embodiment generates a XhoI site at the double underlined LE site and a HindIII site at the double-underlined KL site.

In a preferred embodiment, the presentation structure is a minibody structure. A “minibody” is essentially composed of a minimal antibody complementarity region. The minibody presentation structure generally provides two randomizing regions that in the folded protein are presented along a single face of the tertiary structure. See for example Bianchi et al., J. Mol. Biol. 236(2):649-59 (1994), and references cited therein, all of which are incorporated by reference). Investigators have shown this minimal domain is stable in solution and have used phage selection systems in combinatorial libraries to select minibodies with peptide regions exhibiting high affinity, Kd=10⁻⁷, for the pro-inflammatory cytokine IL-6.

A preferred minibody presentation structure is as follows:

MGRNSQATSGFTFSHEFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRY IVSRDTSQSILYLQKKKGPP (SEQ ID NO:7. The bold, underline regions are the regions which may be randomized. The italized phenylalanine must be invariant in the first randomizing region. The entire peptide is cloned in a three-oligonucleotide variation of the coiled-coil embodiment, thus allowing two different randomizing regions to be incorporated simultaneously. This embodiment utilizes non-palindromic BstXI sites on the termini.

In a preferred embodiment, the presentation structure is a sequence that contains generally two cysteine residues, such that a disulfide bond may be formed, resulting in a conformationally constrained sequence. This embodiment is particularly preferred when secretory targeting sequences are used. As will be appreciated by those in the art, any number of random sequences, with or without spacer or linking sequences, may be flanked with cysteine residues. In other embodiments, effective presentation structures may be generated by the random regions themselves. For example, the random regions may be “doped” with cysteine residues which, under the appropriate redox conditions, may result in highly crosslinked structured conformations, similar to a presentation structure. Similarly, the randomization regions may be controlled to contain a certain number of residues to confer β-sheet or α-helical structures.

In a preferred embodiment, the fusion partner is a targeting sequence. As will be appreciated by those in the art, the localization of proteins within a cell is a simple method for increasing effective concentration and determining function. For example, RAF1 when localized to the mitochondrial membrane can inhibit the anti-apoptotic effect of BCL-2. Similarly, membrane bound Sos induces Ras mediated signaling in T-lymphocytes. These mechanisms are thought to rely on the principle of limiting the search space for ligands, that is to say, the localization of a protein to the plasma membrane limits the search for its ligand to that limited dimensional space near the membrane as opposed to the three dimensional space of the cytoplasm. Alternatively, the concentration of a protein can also be simply increased by nature of the localization. Shuttling the proteins into the nucleus confines them to a smaller space thereby increasing concentration. Finally, the ligand or target may simply be localized to a specific compartment, and inhibitors must be localized appropriately.

Thus, suitable targeting sequences include, but are not limited to, binding sequences capable of causing binding of the expression product to a predetermined molecule or class of molecules while retaining bioactivity of the expression product, (for example by using enzyme inhibitor or substrate sequences to target a class of relevant enzymes); sequences signalling selective degradation, of itself or co-bound proteins; and signal sequences capable of constitutively localizing the candidate expression products to a predetermined cellular locale, including a) subcellular locations such as the Golgi, endoplasmic reticulum, nucleus, nucleoli, nuclear membrane, mitochondria, chloroplast, secretory vesicles, lysosome, and cellular membrane; and b) extracellular locations via a secretory signal. Particularly preferred is localization to either subcellular locations or to the outside of the cell via secretion.

In a preferred embodiment, the targeting sequence is a nuclear localization signal (NLS). NLSs are generally short, positively charged (basic) domains that serve to direct the entire protein in which they occur to the cell's nucleus. Numerous NLS amino acid sequences have been reported including single basic NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro Lys Lys Lys Arg Lys Val (SEQ ID NO:8), Kalderon (1984), et al., Cell, 39:499-509; the human retinoic acid receptor-β nuclear localization signal (ARRRRP) (SEQ ID NO:9); NFκB p50 (EEVQRKRQKL (SEQ ID NO:10); Ghosh et al., Cell 62:1019 (1990); NFκB p65 (EEKRKRTYE(SEQ ID NO:11); Nolan et al., Cell 64:961 (1991); and others (see for example Boulikas, J. Cell. Biochem. 55(1):32-58 (1994), hereby incorporated by reference) and double basic NLS's exemplified by that of the Xenopus (African clawed toad) protein, nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Leu Asp) (SEQ ID NO:12, Dingwall, et al., Cell, 30:449-458, 1982 and Dingwall, et al., J. Cell Biol., 107:641-849; 1988). Numerous localization studies have demonstrated that NLSs incorporated in synthetic peptides or grafted onto selectable proteins not normally targeted to the cell nucleus cause these peptides and selectable proteins to be concentrated in the nucleus. See, for example, Dingwall, and Laskey, Ann, Rev. Cell Biol., 2:367-390, 1986; Bonnerot, et al., Proc. Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo, et al., Proc. Natl. Acad. Sci. USA, 87:458-462, 1990.

In a preferred embodiment, the targeting sequence is a membrane anchoring signal sequence. This is particularly useful since many parasites and pathogens bind to the membrane, in addition to the fact that many intracellular events originate at the plasma membrane. Thus, membrane-bound peptide libraries are useful for both the identification of important elements in these processes as well as for the discovery of effective inhibitors. The invention provides methods for presenting the randomized expression product extracellularly or in the cytoplasmic space. For extracellular presentation, a membrane anchoring region is provided at the carboxyl terminus of the peptide presentation structure. The randomized epression product region is expressed on the cell surface and presented to the extracellular space, such that it can bind to other surface molecules (affecting their function) or molecules present in the extracellular medium. The binding of such molecules could confer function on the cells expressing a peptide that binds the molecule. The cytoplasmic region could be neutral or could contain a domain that, when the extracellular randomized expression product region is bound, confers a function on the cells (activation of a kinase, phosphatase, binding of other cellular components to effect function). Similarly, the randomized expression product-containing region could be contained within a cytoplasmic region, and the transmembrane region and extracellular region remain constant or have a defined function.

Membrane-anchoring sequences are well known in the art and are based on the genetic geometry of mammalian transmembrane molecules. Peptides are inserted into the membrane based on a signal sequence (designated herein as ssTM) and require a hydrophobic transmembrane domain (herein TM). The transmembrane proteins are inserted into the membrane such that the regions encoded 5′ of the transmembrane domain are extracellular and the sequences 3′ become intracellular. Of course, if these transmembrane domains are placed 5′ of the variable region, they will serve to anchor it as an intracellular domain, which may be desirable in some embodiments. ssTMs and TMs are known for a wide variety of membrane bound proteins, and these sequences may be used accordingly, either as pairs from a particular protein or with each component being taken from a different protein, or alternatively, the sequences may be synthetic, and derived entirely from consensus as artificial delivery domains.

As will be appreciated by those in the art, membrane-anchoring sequences, including both ssTM and TM, are known for a wide variety of proteins and any of these may be used. Particularly preferred membrane-anchoring sequences include, but are not limited to, those derived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.

Useful sequences include sequences from: 1) class I integral membrane proteins such as IL-2 receptor beta-chain (residues 1-26 are the signal sequence, 241-265 are the transmembrane residues; see Hatakeyama et al., Science 244:551 (1989) and von Heijne et al, Eur. J. Biochem. 174:671 (1988)) and insulin receptor beta chain (residues 1-27 are the signal, 957-959 are the transmembrane domain and 960-1382 are the cytoplasmic domain; see Hatakeyama, supra, and Ebina et al., Cell 40:747 (1985)); 2) class II integral membrane proteins such as neutral endopeptidase (residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmic domain; see Malfroy et al., Biochem. Biophys. Res. Commun. 144:59 (1987)); 3) type III proteins such as human cytochrome P450 NF25 (Hatakeyama, supra); and 4) type IV proteins such as human P-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 and ICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at the extreme 5′ end of the transcript. These consist of the amino acids 1-32 in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP (SEQ ID NO:13); Nakauchi et al., PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2 (MSSFGYRTLTVALFTLICCPG (SEQ ID NO:14); Staunton et al., Nature (London) 339:61 (1989)). These leader sequences deliver the construct to the membrane while the hydrophobic transmembrane domains, placed 3′ of the random candidate region, serve to anchor the construct in the membrane. These transmembrane domains are encompassed by amino acids 145-195 from CD8 (PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR (SEQ ID NO:15); Nakauchi, supra) and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR (SEQ ID NO:16); Staunton, supra).

Alternatively, membrane anchoring sequences include the GPI anchor, which results in a covalent bond between the molecule and the lipid bilayer via a glycosyl-phosphatidylinositol bond for example in DAF (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO:17), with the bolded serine the site of the anchor; see Homans et al., Nature 333(6170):269-72 (1988), and Moran et al., J. Biol. Chem. 266:1250 (1991)). In order to do this, the GPI sequence from Thy-1 can be cassetted 3′ of the variable region in place of a transmembrane sequence.

Similarly, myristylation sequences can serve as membrane anchoring sequences. It is known that the myristylation of c-src recruits it to the plasma membrane. This is a simple and effective method of membrane localization, given that the first 14 amino acids of the protein are solely responsible for this function: MGSSKSKPKDPSQR (SEQ ID NO:18) (see Cross et al., Mol. Cell. Biol. 4(9):1834 (1984); Spencer et al., Science 262:1019-1024 (1993), both of which are hereby incorporated by reference). This motif has already been shown to be effective in the localization of selectable genes and can be used to anchor the zeta chain of the TCR. This motif is placed 5′ of the variable region in order to localize the construct to the plasma membrane. Other modifications such as palmitoylation can be used to anchor constructs in the plasma membrane; for example, palmitoylation sequences from the G protein-coupled receptor kinase GRK6 sequence (LLQRLFSRQDCCGNCSDSEEELPTRL, (SEQ ID NO:19) with the bold cysteines being palmitolyated; Stoffel et al., J. Biol. Chem 269:27791 (1994)); from rhodopsin (KQFRNCMLTSLCCGKNPLGD (SEQ ID NO:20); Barnstable et al., J. Mol. Neurosci. 5(3):207 (1994)); and the p21 H-ras 1 protein (LNPPDESGPGCMSCKCVLS; (SEQ ID NO:21) Capon et al., Nature 302:33 (1983)).

In a preferred embodiment, the targeting sequence is a lysozomal targeting sequence, including, for example, a lysosomal degradation sequence such as Lamp-2 (KFERQ (SEQ ID NO:22); Dice, Ann. N.Y. Acad. Sci. 674:58 (1992); or lysosomal membrane sequences from Lamp-1 (MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI (SEQ ID NO:23), Uthayakumar et al., Cell. Mol. Biol. Res. 41:405 (1995)) or Lamp-2 (LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF (SEQ ID NO:24), Konecki et la., Biochem. Biophys. Res. Comm. 205:1-5 (1994), both of which show the transmembrane domains in italics and the cytoplasmic targeting signal underlined).

Alternatively, the targeting sequence may be a mitrochondrial localization sequence, including mitochondrial matrix sequences (e.g. yeast alcohol dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST (SEQ ID NO:25); Schatz, Eur. J. Biochem. 165:1-6 (1987)); mitochondrial inner membrane sequences (yeast cytochrome c oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO:26); Schatz, supra); mitochondrial intermembrane space sequences (yeast cytochrome cl; MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITAST LLYADSLTAEAMTA (SEQ ID NO:27); Schatz, supra) or mitochondrial outer membrane sequences (yeast 70 kD outer membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK (SEQ ID NO:28); Schatz, supra).

The target sequences may also be endoplasmic reticulum sequences, including the sequences from calreticulin (KDEL (SEQ ID NO:29); Pelham, Royal Society London Transactions B; 1-10 (1992)) or adenovirus E3/19K protein (LYLSRRSFIDEKKMP (SEQ ID NO:30); Jackson et al., EMBO J. 9:3153 (1990).

Furthermore, targeting sequences also include peroxisome sequences (for example, the peroxisome matrix sequence from Luciferase; SKL (SEQ ID NO:32); Keller et al., PNAS USA 4:3264 (1987)); farnesylation sequences (for example, P21 H-ras 1; LNPPDESGPGCMSCKCVLS (SEQ ID NO:31), with the bold cysteine farnesylated; Capon, supra); geranylgeranylation sequences (for example, protein rab-5A; LTEPTQPTRNQCCSN (SEQ ID NO:33), with the bold cysteines geranylgeranylated; Farnsworth, PNAS USA 91:11963 (1994)); or destruction sequences (cyclin B1; RTALGDIGN (SEQ ID NO:34); Klotzbucher et al., EMBO J. 1:3053 (1996)).

In a preferred embodiment, the targeting sequence is a secretory signal sequence capable of effecting the secretion of the candidate agent. There are a large number of known secretory signal sequences which are placed 5′ to the variable peptide region, and are cleaved from the peptide region to effect secretion into the extracellular space. Secretory signal sequences and their transferability to unrelated proteins are well known, e.g., Silhavy, et al. (1985) Microbiol. Rev. 49, 398-418. This is particularly useful to generate a peptide capable of binding to the surface of, or affecting the physiology of, a target cell that is other than the host cell, e.g., the cell infected with the retrovirus. In a preferred approach, a fusion product is configured to contain, in series, secretion signal peptide-presentation structure-randomized expression product region-presentation structure. In this manner, target cells grown in the vicinity of cells caused to express the library of peptides, are bathed in secreted peptide. Target cells exhibiting a physiological change in response to the presence of a peptide, e.g., by the peptide binding to a surface receptor or by being internalized and binding to intracellular targets, and the secreting cells are localized by any of a variety of selection schemes and the peptide causing the effect determined. Exemplary effects include variously that of a designer cytokine (i.e., a stem cell factor capable of causing hematopoietic stem cells to divide and maintain their totipotential), a factor causing cancer cells to undergo spontaneous apoptosis, a factor that binds to the cell surface of target cells and labels them specifically, etc. Suitable secretory sequences are known, including signals from IL-2 (MYRMQLLSCIALSLALVTNS (SEQ ID NO:35); Villinger et al., J. Immunol. 155:3946 (1995)), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT (SEQ ID NO:36); Roskam et al., Nucleic Acids Res. 7:30 (1979)); preproinsulin (MALWMRLLPLLALLALWGPDPAAAFVN (SEQ ID NO:37); Bell et al., Nature 284:26 (1980)); and influenza HA protein (MKAKLLVLLYAFVAGDQI (SEQ ID NO:38); Sekiwawa et al., PNAS 80:3563)), with cleavage between the non-underlined-underlined junction. A particularly preferred secretory signal sequence is the signal leader sequence from the secreted cytokine IL-4, which comprises the first 24 amino acids of IL-4 as follows: MGLTSQLLPPLFFLLACAGNFVHG (SEQ ID NO:39).

In a preferred embodiment, the fusion partner is a rescue sequence. A rescue sequence is a sequence which may be used to purify or isolate either the candidate agent or the heterologous nucleic acid encoding it. Thus, for example, peptide rescue sequences include purification sequences such as the His₆ tag for use with Ni affinity columns and epitope tags for detection, immunoprecipitation or FACS (fluoroscence-activated cell sorting). Suitable epitope tags include myc (for use with the commercially available 9E10 antibody), the BSP biotinylation target sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST.

Alternatively, the rescue sequence may be a unique oligonucleotide sequence which serves as a probe target site to allow the quick and easy isolation of the retroviral construct, via PCR, related techniques, or hybridization.

In a preferred embodiment, the fusion partner is a stability sequence to confer stability to the candidate bioactive agent or the heterologous nucleic acid encoding it. Thus, for example, peptides may be stabilized by the incorporation of glycines after the initiation methionine (MG or MGGO), for protection of the peptide to ubiquitination as per Varshavsky's N-End Rule, thus conferring long half-life in the cytoplasm. Similarly, two prolines at the C-terminus impart peptides that are largely resistant to carboxypeptidase action. The presence of two glycines prior to the prolines impart both flexibility and prevent structure initiating events in the di-proline to be propagated into the candidate peptide structure. Thus, preferred stability sequences are as follows: MG(X)_(n)GGPP (SEQ ID NO:40), where X is any amino acid and n is an integer of at least four.

In one embodiment, the fusion partner is a dimerization sequence. A dimerization sequence allows the non-covalent association of one random peptide to another random peptide, with sufficient affinity to remain associated under normal physiological conditions. This effectively allows small libraries of random peptides (for example, 10⁴) to become large libraries if two peptides per cell are generated which then dimerize, to form an effective library of 10⁸ (10⁴×10⁴). It also allows the formation of longer random peptides, if needed, or more structurally complex random peptide molecules. The dimers may be homo- or heterodimers.

Dimerization sequences may be a single sequence that self-aggregates, or two sequences, each of which is generated in a different retroviral construct. That is, nucleic acids encoding both a first random peptide with dimerization sequence 1, and a second random peptide with dimerization sequence 2, such that upon introduction into a cell and expression of the nucleic acid, dimerization sequence 1 associates with dimerization sequence 2 to form a new random peptide structure.

Suitable dimerization sequences will encompass a wide variety of sequences. Any number of protein-protein interaction sites are known. In addition, dimerization sequences may also be elucidated using standard methods such as the yeast two hybrid system, traditional biochemical affinity binding studies, or even using the present methods.

The fusion partners may be placed anywhere (i.e. N-terminal, C-terminal, internal) in the structure as the biology and activity permits.

In a preferred embodiment, the fusion partner includes a linker or tethering sequence, as generally described in PCT US 97/01019, that can allow the candidate agents to interact with potential targets unhindered. For example, when the candidate bioactive agent is a peptide, useful linkers include glycine-serine polymers (including, for example, (GS)_(n) (SEQ ID NO:41), (GSGGS)_(n) (SEQ ID NO:42) and (GGGS)_(n), (SEQ ID NO:43) where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art. Glycine-serine polymers are preferred since both of these amino acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Secondly, serine is hydrophilic and therefore able to solubilize what could be a globular glycine chain. Third, similar chains have been shown to be effective in joining subunits of recombinant proteins such as single chain antibodies.

In addition, the fusion partners, including presentation structures, may be modified, randomized, and/or matured to alter the presentation orientation of the randomized expression product. For example, determinants at the base of the loop may be modified to slightly modify the internal loop peptide tertiary structure, which maintaining the randomized amino acid sequence.

Thus, heterologous nucleic acids can be sequences which have not been manipulated in any way, or alternatively, they can be constructed to have fusion partners. In either case, they can be inserted into the shuttle vectors by conventional methods such as enzymatic manipulation and ligation, or preferably, are inserted into the shuttle vector by homologous recombination as described herein.

It is understood by the skilled artisan that while various options (of compounds, properties selected or order of steps) are provided herein, the options are also each provided individually, and can each be individually segregated from the other options provided herein. Moreover, steps which are obvious and known in the art are intended to be within the scope of this invention. For example, there may be additionally washing steps, segregation, or isolation steps. Moreover, additional components to vectors, particularly regulatory elements, cells, cell media, etc., which are routine and known in the art can be incorporated herein without deviating from the spirit and scope of the invention.

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are expressly incorporated by reference in their entirety.

EXAMPLES Example 1

Mammalian cells transfected with a shuttle vector show expression

A shuttle vector (pPYC-R) was constructed in accordance with the schematic shown in FIG. 1. The sequence is provided in FIG. 2. The vector has an IRES at positions 6001-6505, a GFP at 6506-7258, Amp^(R) at 9888-655, an E. coli replication origin at 656-1456, a yeast 2 μ replication origin at 1461-2808, Trp at 3344-4018 and a CMV promoter at 4853-5614 of SEQ ID NO:1 shown in FIG. 2.

1 μg of pPYC-R plasmid was transfected into 30% confluent 293 (Phoenix) cells by a standard Ca²⁺ Phosphate transfection method known in the art to test expression of GFP. After incubation for 48 hours in 37° C. CO₂ incubator, cells transfected by pPYC-R show green fluorescence color under UV microscope as depicted in FIG. 3.

Example 2

Use of yeast to construct shuttle vector with insert, expression of insert

This example demonstrates the in-frame fusion of Rip cDNA, an apoptosis-inducing gene when over-expressed in mammalian cells, to a hemagglutinin (HA) tag in pPYC by recombination with non-virus based vector. Rip is further described in Hsu, et al., Immunity, 4:387-396 (1996), incorporated herein by reference.

1 μg of pPYC plasmid (FIGS. 4 and 5) was cut by EcoR I to linearize and was purified from agarose gel. Rip cDNA was amplified by PCR and was purified from agarose gel. The oligo-nucleotide sequences used to amplify Rip were

ACGACTCACTATAGGCTAGCCGCCACCATGGCTIACCCATACGATGTTC CAGATTACGCTGGGCAACCAGACATGTCCTTGAA (SEQ ID NO:3) and

TTGCCAAAAGACGGCAATATGGTGGAAAATAACGTGTGACTCTAGAG GTACCACGCGTGTTAGTTCTGGCTGACGTAAA (SEQ ID NO:4).

Flanking sequences required for homologous recombination between PCR fragment and vector are underlined. The purified vector and PCR fragment was co-transfected into yeast by a standard Li/PEG method known in the art. Transformants were plated on SD-W selection plate and were incubated in 30° C. incubator for 4 days. Colonies were harvested and pooled together for plasmid mini-preparation to recover recombinant plasmid from yeast. The plasmid from mini-preparation was transformed into E. coli to isolate single colony on LB plus 50 μg/ml ampicilin. Five colonies were picked to grow up for plasmid mini-preparation and subsequent restriction enzyme digestion and sequencing verification.

Clones with Rip cDNA inserted in-frame downstream of the tag (HA) were co-transfected with pGDB, an apoptosis reporter vector, into 30% confluent mammalian 293 (Phoenix) cells by Ca²⁺ phosphate transfection method to test expression of Rip. FIG. 6 shows the results, extensive cell death due to the expression of HA tagged Rip.

Example 3

Mammalian cells transfected with a shuttle vector show expression

A shuttle vector (pCRU5YMS) was constructed in accordance with the schematic shown in FIG. 7. The sequence is provided in FIG. 8.

One microgram of pCRU5YMS was used to transfect virus packaging cell line Phoenix (293) by a standard CaPO₄ transfection method. Under a UV microscope, green fluorescence can be observed after 24 hours, indicating that the GFP has been expressed by pCRU5 promoter. After 48 hours incubation at 30° C., medium containing newly packaged viruses was harvested for a second round of infection of Hela cells. 48 hours after infection, green fluorescence can be observed in most of the Hela cells under the UV microscope as shown in FIG. 9.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 43 <210> SEQ ID NO 1 <211> LENGTH: 10100 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: constructed vectors <400> SEQUENCE: 1 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg 60 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 120 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 180 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 240 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 300 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 360 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 420 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 480 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 540 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 600 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 660 attggtaact gtcagaccaa gtttactcat atatacttta gattgatttg cggccgcaaa 720 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 780 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 840 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 900 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 960 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 1020 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 1080 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 1140 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 1200 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 1260 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 1320 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 1380 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 1440 agcaacgcgg ccttttgctc gaacgaagca tctgtgcttc attttgtaga acaaaaatgc 1500 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa 1560 tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa 1620 aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac 1680 agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct 1740 acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc 1800 tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag 1860 gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact 1920 tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc 1980 ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt 2040 tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac 2100 tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt 2160 actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg 2220 agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca 2280 cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata 2340 ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc 2400 gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata ggaacttcgg 2460 aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg 2520 cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta tatatatata 2580 catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat gcgtctattt 2640 atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca tgcggggtat 2700 cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc ctcaattgga 2760 ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatatta agaaaccatt 2820 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 2880 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 2940 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 3000 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccataga 3060 tcaacgacat tactatatat ataatatagg aagcatttaa tagacagcat cgtaatatat 3120 gtgtactttg cagttatgac gccagatggc agtagtggaa gatattcttt attgaaaaat 3180 agcttgtcac cttacgtaca atcttgatcc ggagcttttc tttttttgcc gattaagaat 3240 taattcggtc gaaaaaagaa aaggagaggg ccaagaggga gggcattggt gactattgag 3300 cacgtgagta tacgtgatta agcacacaaa ggcagcttgg agtatgtctg ttattaattt 3360 cacaggtagt tctggtccat tggtgaaagt ttgcggcttg cagagcacag aggccgcaga 3420 atgtgctcta gattccgatg ctgacttgct gggtattata tgtgtgccca atagaaagag 3480 aacaattgac ccggttattg caaggaaaat ttcaagtctt gtaaaagcat ataaaaatag 3540 ttcaggcact ccgaaatact tggttggcgt gtttcgtaat caacctaagg aggatgtttt 3600 ggctctggtc aatgattacg gcattgatat cgtccaactg catggagatg agtcgtggca 3660 agaataccaa gagttcctcg gtttgccagt tattaaaaga ctcgtatttc caaaagactg 3720 caacatacta ctcagtgcag cttcacagaa acctcattcg tttattccct tgtttgattc 3780 agaagcaggt gggacaggtg aacttttgga ttggaactcg atttctgact gggttggaag 3840 gcaagagagc cccgaaagct tacattttat gttagctggt ggactgacgc cagaaaatgt 3900 tggtgatgcg cttagattaa atggcgttat tggtgttgat gtaagcggag gtgtggagac 3960 aaatggtgta aaagactcta acaaaatagc aaatttcgtc aaaaatgcta agaaataggt 4020 tattactgag tagtatttat ttaagtattg tttgtgcact tgccgatcac tatggccatt 4080 taatgtaaat acttaagaaa aaaaaccaaa ttaattttga tacatgctgc atgtgaagac 4140 ccccgctgac gggtagtcaa tcactcagag gagaccctcc caaggcagcg agaccacaag 4200 tcggaaatga aagacccccg ctgacgggta gtcaatcact cagaggagac cctcccaagg 4260 aacagcgaga ccacaagtcg gatgcaactg caagagggtt tattggatac acgggtaccc 4320 gggcgactca gtcaatcgga ggactggcgc cccgagtgag gggttgtggg ctcttttatt 4380 gagctcgggg agcagaagcg cgcgaacaga agcgagaagc gaactgattg gttagttcaa 4440 ataaggcaca gggtcatttc aggtccttgg ggcaccctgg aaacatctga tggttctcta 4500 gaaactgctg agggctggac cgcatctggg gaccatctgt tcttggccct gagccggggc 4560 aggaactgct taccacagat atcctgtttg gcccatattc agctgttcca tctgttcttg 4620 gccctgagcc ggggcaggaa ctgcttacca cagatatcct gtttggccca tattcagctg 4680 ttccatctgt tcctgacctt gatctgaact tctctattct cagttatgta tttttccatg 4740 ccttgcaaaa tggcgttact taagctagct tgccaaacct acaggtgggg tctttcattc 4800 cccccttttt ctggagacta aataaaatct tttattttat cgtcgatcga ctagatcttc 4860 aatattggcc attagccata ttattcattg gttatatagc ataaatcaat attggctatt 4920 ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc tcatgtccaa 4980 tatgaccgcc atgttggcat tgattattga ctagttatta atagtaatca attacggggt 5040 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 5100 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 5160 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 5220 acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg 5280 gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc 5340 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacacca 5400 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 5460 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactgcg 5520 atcgcccgcc ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag 5580 cagagctcgt ttagtgaacc gtcagatcac tagaagcttt attgcggtag tttatcacag 5640 ttaaattgct aacgcagtca gtgcttctga cacaacagtc tcgaacttaa gctgcagtga 5700 ctctcttaag gtagccttgc agaagttggt cgtgaggcac tgggcaggta agtatcaagg 5760 ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag agaagactct 5820 tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacag 5880 gtgtccactc ccagttcaat tacagctctt aaggctagag tacttaatac gactcactat 5940 aggctagcct cgagccgcca ccatggaatt cacgtgcatg caggccttaa ttaagtcgac 6000 acgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct 6060 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg 6120 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta 6180 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag 6240 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg 6300 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat 6360 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca 6420 tgtgtttagt cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc 6480 tttgaaaaac acgatgataa tatgggggat ccaccggtcg ccaccatggt gagcaagggc 6540 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 6600 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 6660 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 6720 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 6780 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 6840 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 6900 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 6960 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 7020 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 7080 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 7140 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 7200 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgctcgacg 7260 ataaattccc tttagtgagg gttaatgctt cgagcagaca tgataagata cattgatgag 7320 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ctatttgtga aatttgtgat 7380 gctattgctg tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc 7440 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac 7500 ctctacaaat gtggtaaaat ccgataagga tccggcagtc tagaggatgg tccacccccg 7560 gggtcggcag ccttcacgtg ggcggcgtgt atccaagctg cgatgccgtc tactttgagg 7620 gcggtggggg tggtcagcag gactgtgtaa ggtcctttcc agcgaggttc taggttctta 7680 gtctggtgtc ggcggaccca cactgtgtcg ccgactcggt aagggtgagg taccaccggt 7740 cggtccagtt gttcttggta cgtgccgcca gaggtctcca gacttcgtgc tggactaagt 7800 agagagcctg taagtgagct tggagagagg ggctgttagt aactcttgtc atgtcagggt 7860 cagggaagtt tacaaggggc gggggtgccc catataagat ctcatatggc catatggggg 7920 cgcctagaga aggagtgagg gctggataaa gggaggatcg aggcggggtc gaacgaggag 7980 gttcaagggg gagagacggg gcggatggag gaagaggagg cggaggctta gggtgtacaa 8040 agggcttgac ccagggaggg gggtcaaaag ccaaggcttc ccaggtcacg atgtagggga 8100 cctggtctgg gtgtccatgc gggccaggtg aaaagacctt gatcttaacc tgggtgatga 8160 ggtctcggtt aaaggtgccg tctcgcggcc atccgacgtt aaaggttggc cattctgcag 8220 agcagaaggt aacccaacgt ctcttcttga catctaccga ctggttgtga gcgatccgct 8280 cgacatcttt ccagtgacct aaggtcaaac ttaagggagt ggtaacagtc tggcccgggc 8340 ccatattttc agacaaatac agaaacacag tcagacagag acaacacaga acgatgctgc 8400 agcagacaag acgcgcggcg cggcttcggt cccaaaccga aagcaaaaat tcagacggag 8460 gcgggaactg ttttaggttc tcgtctccta ccagaaccac atatccctcc tctaaggggg 8520 gtgcaccaaa gagtccaaaa cgatcgggat ttttggactc aggtcgggcc acaaaaacgg 8580 cccccgaagt ccctgggacg tctcccaggg ttgcggccgg gtgttccgaa ctcgtcagtt 8640 ccaccacggg tccgccagat acagagctag ttagctaact agtaccgacg caggcgcata 8700 aaatcagtca tagacactag acaatcggac agacacagat aagttgctgg ccagcttacc 8760 tcccggtggt gggtcggtgg tccctgggca ggggtctccc gatcccggac gagcccccaa 8820 atgaaagacc cccgctgacg ggtagtcaat cactcagagg agaccctccc aaggaacagc 8880 gagaccacaa gtcggatgca actgcaagag ggtttattgg atacacgggt acccgggcga 8940 ctcagtcaat cggaggactg gcgccccgag tgaggggttg tgggctcttt tattgagctc 9000 ggggagcaga agcgcgcgaa cagaagcgag aagcgaactg attggttagt tcaaataagg 9060 cacagggtca tttcaggtcc ttggggcacc ctggaaacat ctgatggttc tctagaaact 9120 gctgagggct ggaccgcatc tggggaccat ctgttcttgg ccctgagccg gggcaggaac 9180 tgcttaccac agatatcctg tttggcccat attcagctgt tccatctgtt cttggccctg 9240 agccggggca ggaactgctt accacagata tccgctttgg cccatattca gctgttccat 9300 ctgttcctga ccttgatctg aacttttcta ttctcagtta tgtatttttc catgccttgc 9360 aaaatggcgt tacttaagct agcttgccaa acctacaggt ggggtctttc acatgtatat 9420 gtcaaaaata aaaatcaact aattgactag taattaatat gactggcata atgggaaatt 9480 gatcctgaca gatgcaaact ggcttctcag cagcgcattt atgttgtcaa ctgaggaagg 9540 aaacgttaat gacagaaact ctaagtaatt tccacgttta tctattttta tttatactag 9600 ctttggtaac aggaatattg cagcattcat gcacattgaa acccttatga aataaaaaca 9660 tctgtgcatt taaaatggaa ttaacatttt aaatgttaaa aaaagctggc ttagcttccc 9720 cccgccccct agggcataga acaagtcaaa tgctttatat atttgagttt gggatgtatt 9780 aggaaactcc taagagcaaa gctgttcttg aagacgaaag ggcctcgtga tacgcctatt 9840 tttataggtt aatgtcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 9900 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 9960 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 10020 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 10080 tcgccccgaa gaacgttttc 10100 <210> SEQ ID NO 2 <211> LENGTH: 9687 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: constructed vectors <400> SEQUENCE: 2 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg 660 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 720 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac 780 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt 840 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa 900 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact 960 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac 1020 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact 1080 ataggctagc cgccaccatg gcttacccat acgatgttcc agattacgct gggcaaccag 1140 acatgtcctt gaatgtcatt aagatgaaat ccagtgactt cctggagagt gcagaactgg 1200 acagcggagg ctttgggaag gtgtctctgt gtttccacag aacccaggga ctcatgatca 1260 tgaaaacagt gtacaagggg cccaactgca ttgagcacaa cgaggccctc ttggaggagg 1320 cgaagatgat gaacagactg agacacagcc gggtggtgaa gctcctgggc gtcatcatag 1380 aggaagggaa gtactccctg gtgatggagt acatggagaa gggcaacctg atgcacgtgc 1440 tgaaagccga gatgagtact ccgctttctg taaaaggaag gataatttgg gaaatcattg 1500 aaggaatgtg ctacttacat gaaaaggcgt gatacacaag gacctgaagc ctgaaaatat 1560 ccttgttgat aatgacttcc acattaagat cgcagacctc ggccttgcct cctttaagat 1620 gtggagcaaa ctgaataatg aagagcacaa tgagctgagg gaagtggacg gcaccgctaa 1680 gaagaatggc ggcaccctct actacatggc gcccgagcac ctgaatgacg tcaacgcaaa 1740 gcccacagag aagtcggatg tgtacagctt tgctgtagta ctctgggcga tatttgcaaa 1800 taaggagcca tatgaaaatg ctatctgtga gcagcagttg ataatgtgca taaaatctgg 1860 gaacaggcca gatgtggatg acatcactga gtactgccca agagaaatta tcagtctcat 1920 gaagctctgc tgggaagcga atccggaagc tcggccgaca tttcctggca ttgaagaaaa 1980 atttaggcct ttttatttaa gtcaattaga agaaagtgta gaagaggacg tgaagagttt 2040 aaagaaagag tattcaaacg aaaatgcagt tgtgaagaga atgcagtctc ttcaacttga 2100 ttgtgtggca gtaccttcaa gccggtcaaa ttcagccaca gaacagcctg gttcactgca 2160 cagttcccag ggacttggga tgggtcctgt ggaggagtcc tggtttgctc cttccctgga 2220 gcacccacaa gaagagaatg agcccagcct gcagagtaaa ctccaagacg aagccaacta 2280 ccatctttat ggcagccgca tggacaggca gacgaaacag cagcccagac agaatgtggc 2340 ttacaacaga gaggaggaaa ggagacgcag ggtctcccat gacccttttg cacagcaaag 2400 accttacgag aattttcaga atacagaggg aaaaggcact gtttattcca gtgcagccag 2460 tcatggtaat gcagtgcacc agccctcagg gctcaccagc caacctcaag tactgtatca 2520 gaacaatgga ttatatagct cacatggctt tggaacaaga ccactggatc caggaacagc 2580 aggtcccaga gtttggtaca ggccaattcc aagtcatatg cctagtctgc ataatatccc 2640 agtgcctgag accaactatc taggaaatac acccaccatg ccattcagct ccttgccacc 2700 aacagatgaa tctataaaat ataccatata caatagtact ggcattcaga ttggagccta 2760 caattatatg gagattggtg ggacgagttc atcactacta gacagcacaa atacgaactt 2820 caaagaagag ccagctgcta agtaccaagc tatctttgat aataccacta gtctgacgga 2880 taaacacctg gacccaatca gggaaaatct gggaaagcac tggaaaaact gtgcccgtaa 2940 actgggcttc acacagtctc agattgatga aattgaccat gactatgagc gagatggact 3000 gaaagaaaag gtttaccaga tgctccaaaa gtgggtgatg agggaaggca taaagggagc 3060 cacggtgggg aagctggccc aggcgctcca ccagtgttcc aggatcgacc ttctgagcag 3120 cttgatttac gtcagccaga actaacacgc gtggtacctc tagagtcgac acgttatttt 3180 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga 3240 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg 3300 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt 3360 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat 3420 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg 3480 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg 3540 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt 3600 cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 3660 acgatgataa tatgggggat ccaccggtcg ccaccatggt gagcaagggc gaggagctgt 3720 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca 3780 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct 3840 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg 3900 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca 3960 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga 4020 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca 4080 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc 4140 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc 4200 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca 4260 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga 4320 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg 4380 ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcttccct ttagtgaggg 4440 ttaatgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga 4500 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 4560 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 4620 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc 4680 cgataaggat cgatccgggc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 4740 aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc 4800 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 4860 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 4920 tcgggggctc cctttagggt tccgatttag agctttacgg cacctcgacc gcaaaaaact 4980 tgatttgggt gatgctcgaa cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac 5040 gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc 5100 aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat 5160 gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt acagaacaga 5220 aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt ttgttctaca 5280 aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact ttttttctcc 5340 tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc cgttaaggtt 5400 agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga ctccacttcc 5460 cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa ggcatccccg 5520 attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg atagcgttga 5580 tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc tatatactac 5640 gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat agttcttact 5700 acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta gaggtcgagt 5760 ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga tatagcacag 5820 agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt cgcaatattt 5880 tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtct tcagagcgct 5940 tttggttttc aaaagcgctc tgaagttcct atactttcta gagaatagga acttcggaat 6000 aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac 6060 atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat atatatacat 6120 gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg tctatttatg 6180 taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc ggggtatcgt 6240 atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc aattggatta 6300 gtctcatcct tcaatgctat catttccttt gatattggat catattaaga aaccattatt 6360 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc 6420 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 6480 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 6540 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatagatca 6600 acgacattac tatatatata atataggaag catttaatag acagcatcgt aatatatgtg 6660 tactttgcag ttatgacgcc agatggcagt agtggaagat attctttatt gaaaaatagc 6720 ttgtcacctt acgtacaatc ttgatccgga gcttttcttt ttttgccgat taagaattaa 6780 ttcggtcgaa aaaagaaaag gagagggcca agagggaggg cattggtgac tattgagcac 6840 gtgagtatac gtgattaagc acacaaaggc agcttggagt atgtctgtta ttaatttcac 6900 aggtagttct ggtccattgg tgaaagtttg cggcttgcag agcacagagg ccgcagaatg 6960 tgctctagat tccgatgctg acttgctggg tattatatgt gtgcccaata gaaagagaac 7020 aattgacccg gttattgcaa ggaaaatttc aagtcttgta aaagcatata aaaatagttc 7080 aggcactccg aaatacttgg ttggcgtgtt tcgtaatcaa cctaaggagg atgttttggc 7140 tctggtcaat gattacggca ttgatatcgt ccaactgcat ggagatgagt cgtggcaaga 7200 ataccaagag ttcctcggtt tgccagttat taaaagactc gtatttccaa aagactgcaa 7260 catactactc agtgcagctt cacagaaacc tcattcgttt attcccttgt ttgattcaga 7320 agcaggtggg acaggtgaac ttttggattg gaactcgatt tctgactggg ttggaaggca 7380 agagagcccc gaaagcttac attttatgtt agctggtgga ctgacgccag aaaatgttgg 7440 tgatgcgctt agattaaatg gcgttattgg tgttgatgta agcggaggtg tggagacaaa 7500 tggtgtaaaa gactctaaca aaatagcaaa tttcgtcaaa aatgctaaga aataggttat 7560 tactgagtag tatttattta agtattgttt gtgcacttgc cgatcgcgta tggtgcactc 7620 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 7680 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 7740 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 7800 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 7860 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 7920 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 7980 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 8040 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 8100 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 8160 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 8220 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 8280 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 8340 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 8400 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 8460 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 8520 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 8580 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 8640 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 8700 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 8760 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 8820 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 8880 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 8940 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 9000 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 9060 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 9120 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 9180 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 9240 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 9300 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 9360 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 9420 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 9480 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 9540 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 9600 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 9660 cttttgctca catggctcga cagatct 9687 <210> SEQ ID NO 3 <211> LENGTH: 83 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: PCR primer for RID <400> SEQUENCE: 3 acgactcact ataggctagc cgccaccatg gcttacccat acgatgttcc agattacgct 60 gggcaaccag acatgtcctt gaa 83 <210> SEQ ID NO 4 <211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: PCR primer for RID <400> SEQUENCE: 4 ttgccaaaag acggcaatat ggtggaaaat aacgtgtcga ctctagaggt accacgcgtg 60 ttagttctgg ctgacgtaaa 80 <210> SEQ ID NO 5 <211> LENGTH: 8614 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: constructed vectors <400> SEQUENCE: 5 atcacgaggc cctttcgtct tcaagaacag ctttgctctt aggagtttcc taatacatcc 60 caaactcaaa tatataaagc atttgacttg ttctatgccc tagttattaa tagtaatcaa 120 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 180 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 240 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 300 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 360 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 420 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 480 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 540 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 600 acaactccgc cccattgacg caaatgggcg gtaggcatgt acggtgggag gtctatataa 660 gcagagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact 720 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt 780 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt 840 catttggggg ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg 900 ggaggtaagc tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga 960 ttttatgcgc ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg 1020 tggaactgac gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg 1080 gggccgtttt tgtggcccga cctgagtcca aaaatcccga tcgttttgga ctctttggtg 1140 cacccccctt agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc 1200 cgcctccgtc tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct 1260 gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata 1320 tcggcccggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg 1380 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct 1440 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag 1500 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc 1560 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc 1620 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg 1680 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag 1740 gcgcccccat atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc 1800 ctgaccctga catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc 1860 tctacttagt ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg 1920 accgaccggt ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc 1980 agactaagaa cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca 2040 ccgccctcaa agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg 2100 accccggggg tggaccatcc tctagactgc cggatctcga gggatccacc accatggacc 2160 cccattaaat tggaattcct gcagcccggg ggatccacta gttctagagc gaattaattc 2220 cggttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct 2280 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg 2340 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta 2400 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag 2460 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg 2520 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat 2580 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca 2640 tgtgtttagt cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc 2700 tttgaaaaac acgatgataa tatgggggat ccaccggtcg ccaccatggt gagcaagggc 2760 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 2820 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 2880 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 2940 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 3000 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 3060 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 3120 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 3180 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 3240 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 3300 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 3360 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 3420 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgctcgacg 3480 ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt 3540 aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg 3600 agaatagaga agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac 3660 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct 3720 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 3780 acagatggtc cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt 3840 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc 3900 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc 3960 ctcactcggg gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata 4020 aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg 4080 agtgattgac tacccgtcag cgggggtctt tcatttccga cttgtggtct cgctgccttg 4140 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtcttcacat gcagcatgta 4200 tcaaaattaa tttggttttt tttcttaagt atttacatta aatggccata gtgatcggca 4260 agtgcacaaa caatacttaa ataaatacta ctcagtaata acctatttct tagcattttt 4320 gacgaaattt gctattttgt tagagtcttt tacaccattt gtctccacac ctccgcttac 4380 atcaacacca ataacgccat ttaatctaag cgcatcacca acattttctg gcgtcagtcc 4440 accagctaac ataaaatgta agctttcggg gctctcttgc cttccaaccc agtcagaaat 4500 cgagttccaa tccaaaagtt cacctgtccc acctgcttct gaatcaaaca agggaataaa 4560 cgaatgaggt ttctgtgaag ctgcactgag tagtatgttg cagtcttttg gaaatacgag 4620 tcttttaata actggcaaac cgaggaactc ttggtattct tgccacgact catctccatg 4680 cagttggacg atatcaatgc cgtaatcatt gaccagagcc aaaacatcct ccttaggttg 4740 attacgaaac acgccaacca agtatttcgg agtgcctgaa ctatttttat atgcttttac 4800 aagacttgaa attttccttg caataaccgg gtcaattgtt ctctttctat tgggcacaca 4860 tataataccc agcaagtcag catcggaatc tagagcacat tctgcggcct ctgtgctctg 4920 caagccgcaa actttcacca atggaccaga actacctgtg aaattaataa cagacatact 4980 ccaagctgcc tttgtgtgct taatcacgta tactcacgtg ctcaatagtc accaatgccc 5040 tccctcttgg ccctctcctt ttcttttttc gaccgaatta attcttaatc ggcaaaaaaa 5100 gaaaagctcc ggatcaagat tgtacgtaag gtgacaagct atttttcaat aaagaatatc 5160 ttccactact gccatctggc gtcataactg caaagtacac atatattacg atgctgtcta 5220 ttaaatgctt cctatattat atatatagta atgtcgttga tctatggtgc actctcagta 5280 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 5340 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 5400 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc 5460 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct taatatgatc 5520 caatatcaaa ggaaatgata gcattgaagg atgagactaa tccaattgag gagtggcagc 5580 atatagaaca gctaaagggt agtgctgaag gaagcatacg ataccccgca tggaatggga 5640 taatatcaca ggaggtacta gactaccttt catcctacat aaatagacgc atataagtac 5700 gcatttaagc ataaacacgc actatgccgt tcttctcatg tatatatata tacaggcaac 5760 acgcagatat aggtgcgacg tgaacagtga gctgtatgtg cgcagctcgc gttgcatttt 5820 cggaagcgct cgttttcgga aacgctttga agttcctatt ccgaagttcc tattctctag 5880 aaagtatagg aacttcagag cgcttttgaa aaccaaaagc gctctgaaga cgcactttca 5940 aaaaaccaaa aacgcaccgg actgtaacga gctactaaaa tattgcgaat accgcttcca 6000 caaacattgc tcaaaagtat ctctttgcta tatatctctg tgctatatcc ctatataacc 6060 tacccatcca cctttcgctc cttgaacttg catctaaact cgacctctac attttttatg 6120 tttatctcta gtattactct ttagacaaaa aaattgtagt aagaactatt catagagtga 6180 atcgaaaaca atacgaaaat gtaaacattt cctatacgta gtatatagag acaaaataga 6240 agaaaccgtt cataattttc tgaccaatga agaatcatca acgctatcac tttctgttca 6300 caaagtatgc gcaatccaca tcggtataga atataatcgg ggatgccttt atcttgaaaa 6360 aatgcacccg cagcttcgct agtaatcagt aaacgcggga agtggagtca ggcttttttt 6420 atggaagaga aaatagacac caaagtagcc ttcttctaac cttaacggac ctacagtgca 6480 aaaagttatc aagagactgc attatagagc gcacaaagga gaaaaaaagt aatctaagat 6540 gctttgttag aaaaatagcg ctctcgggat gcatttttgt agaacaaaaa agaagtatag 6600 attctttgtt ggtaaaatag cgctctcgcg ttgcatttct gttctgtaaa aatgcagctc 6660 agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttttac aaaaatgaag 6720 cacagattct tcgttggtaa aatagcgctt tcgcgttgca tttctgttct gtaaaaatgc 6780 agctcagatt ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tctacaaaat 6840 gaagcacaga tgcttcgttc gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 6900 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 6960 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 7020 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 7080 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 7140 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 7200 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 7260 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 7320 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 7380 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 7440 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 7500 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 7560 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 7620 aaaatgaagt ttgcgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta 7680 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 7740 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 7800 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 7860 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 7920 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 7980 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 8040 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 8100 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 8160 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 8220 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 8280 ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat 8340 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 8400 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 8460 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 8520 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 8580 ttgtctcatg acattaacct ataaaaatag gcgt 8614 <210> SEQ ID NO 6 <211> LENGTH: 61 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: coiled-coil presentation structure <300> PUBLICATION INFORMATION: <301> AUTHORS: Martin et al., <303> JOURNAL: EMBO J. <304> VOLUME: 13 <305> ISSUE: 22 <306> PAGES: 5303-5309 <307> DATE: 1994 <400> SEQUENCE: 6 Met Gly Cys Ala Ala Leu Glu Ser Glu Val Ser Ala Leu Glu Ser Glu 1 5 10 15 Val Ala Ser Leu Glu Ser Glu Val Ala Ala Leu Gly Arg Gly Asp Met 20 25 30 Pro Leu Ala Ala Val Lys Ser Lys Leu Ser Ala Val Lys Ser Lys Leu 35 40 45 Ala Ser Val Lys Ser Lys Leu Ala Ala Cys Gly Pro Pro 50 55 60 <210> SEQ ID NO 7 <211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: minibody presentation structure <400> SEQUENCE: 7 Met Gly Arg Asn Ser Gln Ala Thr Ser Gly Phe Thr Phe Ser His Phe 1 5 10 15 Tyr Met Glu Trp Val Arg Gly Gly Glu Tyr Ile Ala Ala Ser Arg His 20 25 30 Lys His Asn Lys Tyr Thr Thr Glu Tyr Ser Ala Ser Val Lys Gly Arg 35 40 45 Tyr Thr Ile Val Ser Arg Asp Thr Ser Gln Ser Ile Leu Tyr Leu Gln 50 55 60 Lys Lys Lys Gly Pro Pro 65 70 <210> SEQ ID NO 8 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Monkey virus <300> PUBLICATION INFORMATION: <301> AUTHORS: Kalderon, <303> JOURNAL: Cell <304> VOLUME: 39 <306> PAGES: 499-509 <307> DATE: 1984 <400> SEQUENCE: 8 Pro Lys Lys Lys Arg Lys Val 1 5 <210> SEQ ID NO 9 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9 Ala Arg Arg Arg Arg Pro 1 5 <210> SEQ ID NO 10 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: NLS <300> PUBLICATION INFORMATION: <301> AUTHORS: Ghosh et al., <303> JOURNAL: Cell <304> VOLUME: 62 <306> PAGES: 1019-1019 <307> DATE: 1990 <400> SEQUENCE: 10 Glu Glu Val Gln Arg Lys Arg Gln Lys Leu 1 5 10 <210> SEQ ID NO 11 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: NLS <300> PUBLICATION INFORMATION: <303> JOURNAL: J. Cell. Biochem. <304> VOLUME: 55 <305> ISSUE: 1 <306> PAGES: 32-58 <307> DATE: 1994 <300> PUBLICATION INFORMATION: <301> AUTHORS: Nolan et al., <303> JOURNAL: Cell <304> VOLUME: 64 <306> PAGES: 961-961 <307> DATE: 1991 <400> SEQUENCE: 11 Glu Glu Lys Arg Lys Arg Thr Tyr Glu 1 5 <210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: African clawed toad <300> PUBLICATION INFORMATION: <301> AUTHORS: Dingwall et al., <303> JOURNAL: J. Cell Biol. <304> VOLUME: 30 <306> PAGES: 449-458 <307> DATE: 1988 <300> PUBLICATION INFORMATION: <301> AUTHORS: Dingwall et al., <303> JOURNAL: Cell <304> VOLUME: 30 <306> PAGES: 449-458 <307> DATE: 1982 <400> SEQUENCE: 12 Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys 1 5 10 15 Lys Lys Leu Asp 20 <210> SEQ ID NO 13 <211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: signal sequence <300> PUBLICATION INFORMATION: <301> AUTHORS: Nakauchi et al., <303> JOURNAL: Proc. Natl. Acad. Sci. U.S.A. <304> VOLUME: 82 <306> PAGES: 5126-5126 <307> DATE: 1985 <400> SEQUENCE: 13 Met Ala Ser Pro Leu Thr Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu 1 5 10 15 Gly Glu Ser Ile Leu Gly Ser Gly Glu Ala Lys Pro Gln Ala Pro 20 25 30 <210> SEQ ID NO 14 <211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: signal sequence <300> PUBLICATION INFORMATION: <301> AUTHORS: Staunton et al., <303> JOURNAL: Nature <304> VOLUME: 339 <306> PAGES: 61-61 <307> DATE: 1989 <400> SEQUENCE: 14 Met Ser Ser Phe Gly Tyr Arg Thr Leu Thr Val Ala Leu Phe Thr Leu 1 5 10 15 Ile Cys Cys Pro Gly 20 <210> SEQ ID NO 15 <211> LENGTH: 51 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: transmembrane domains <300> PUBLICATION INFORMATION: <301> AUTHORS: Nakauchi et al., <303> JOURNAL: Proc. Natl. Acad. Sci. U.S.A. <304> VOLUME: 82 <306> PAGES: 5126-5126 <307> DATE: 1985 <400> SEQUENCE: 15 Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr 1 5 10 15 Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 20 25 30 Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr 35 40 45 His Ser Arg 50 <210> SEQ ID NO 16 <211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: transmembrane domain <300> PUBLICATION INFORMATION: <301> AUTHORS: Staunton et al., <303> JOURNAL: Nature <304> VOLUME: 339 <306> PAGES: 61-61 <307> DATE: 1989 <400> SEQUENCE: 16 Met Val Ile Ile Val Thr Val Val Ser Val Leu Leu Ser Leu Phe Val 1 5 10 15 Thr Ser Val Leu Leu Cys Phe Ile Phe Gly Gln His Leu Arg Gln Gln 20 25 30 Arg <210> SEQ ID NO 17 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: anchor site <300> PUBLICATION INFORMATION: <303> JOURNAL: Nature <304> VOLUME: 333 <305> ISSUE: 6170 <306> PAGES: 269-272 <307> DATE: 1988 <300> PUBLICATION INFORMATION: <303> JOURNAL: J. Biol. Chem. <304> VOLUME: 266 <306> PAGES: 1250-1250 <307> DATE: 1991 <400> SEQUENCE: 17 Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser 1 5 10 15 Gly His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr 20 25 30 Met Gly Leu Leu Thr 35 <210> SEQ ID NO 18 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: myristylation sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Mol. Cell. Biol. <304> VOLUME: 4 <305> ISSUE: 9 <306> PAGES: 1834-1834 <307> DATE: 1984 <300> PUBLICATION INFORMATION: <303> JOURNAL: Science <304> VOLUME: 262 <306> PAGES: 1019-1024 <307> DATE: 1993 <400> SEQUENCE: 18 Met Gly Ser Ser Lys Ser Lys Pro Lys Asp Pro Ser Gln Arg 1 5 10 <210> SEQ ID NO 19 <211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: palmitolyated sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: J. Biol. Chem. <304> VOLUME: 269 <306> PAGES: 27791-27791 <307> DATE: 1994 <400> SEQUENCE: 19 Leu Leu Gln Arg Leu Phe Ser Arg Gln Asp Cys Cys Gly Asn Cys Ser 1 5 10 15 Asp Ser Glu Glu Glu Leu Pro Thr Arg Leu 20 25 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: palmitolyated sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: J. Mol. Neurosci. <304> VOLUME: 5 <305> ISSUE: 3 <306> PAGES: 207-207 <307> DATE: 1994 <400> SEQUENCE: 20 Lys Gln Phe Arg Asn Cys Met Leu Thr Ser Leu Cys Cys Gly Lys Asn 1 5 10 15 Pro Leu Gly Asp 20 <210> SEQ ID NO 21 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: pamitolyated sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Nature <304> VOLUME: 302 <306> PAGES: 33-33 <307> DATE: 1983 <400> SEQUENCE: 21 Leu Asn Pro Pro Asp Glu Ser Gly Pro Gly Cys Met Ser Cys Lys Cys 1 5 10 15 Val Leu Ser <210> SEQ ID NO 22 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: lysosomal degradation sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Ann. N. Y. Acad. Sci. <304> VOLUME: 674 <306> PAGES: 58-58 <307> DATE: 1992 <400> SEQUENCE: 22 Lys Phe Glu Arg Gln 1 5 <210> SEQ ID NO 23 <211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: lysosomal membrane sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Cell. Mol. Biol. Res. <304> VOLUME: 41 <306> PAGES: 405-405 <307> DATE: 1995 <400> SEQUENCE: 23 Met Leu Ile Pro Ile Ala Gly Phe Phe Ala Leu Ala Gly Leu Val Leu 1 5 10 15 Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg Lys Arg Ser His Ala Gly 20 25 30 Tyr Gln Thr Ile 35 <210> SEQ ID NO 24 <211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: lysosomal membrane sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Biochem. Biophys. Res. Commun. <304> VOLUME: 205 <306> PAGES: 1-5 <307> DATE: 1994 <400> SEQUENCE: 24 Leu Val Pro Ile Ala Val Gly Ala Ala Leu Ala Gly Val Leu Ile Leu 1 5 10 15 Val Leu Leu Ala Tyr Phe Ile Gly Leu Lys His His His Ala Gly Tyr 20 25 30 Glu Gln Phe 35 <210> SEQ ID NO 25 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: mitrochondrial localization sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Eur. J. Biochem. <304> VOLUME: 165 <306> PAGES: 1-6 <307> DATE: 1987 <400> SEQUENCE: 25 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe Ser Arg Asn Ile Leu Arg Leu Gln Ser Thr 20 25 <210> SEQ ID NO 26 <211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: mictrochondrial localization sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Eur. J. Biochem. <304> VOLUME: 165 <306> PAGES: 1-6 <307> DATE: 1987 <400> SEQUENCE: 26 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15 Thr Leu Cys Ser Ser Arg Tyr Leu Leu 20 25 <210> SEQ ID NO 27 <211> LENGTH: 64 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: mitrochondrial localization sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Eur. J. Biochem. <304> VOLUME: 165 <306> PAGES: 1-6 <307> DATE: 1987 <400> SEQUENCE: 27 Met Phe Ser Met Leu Ser Lys Arg Trp Ala Gln Arg Thr Leu Ser Lys 1 5 10 15 Ser Phe Tyr Ser Thr Ala Thr Gly Ala Ala Ser Lys Ser Gly Lys Leu 20 25 30 Thr Gln Lys Leu Val Thr Ala Gly Val Ala Ala Ala Gly Ile Thr Ala 35 40 45 Ser Thr Leu Leu Tyr Ala Asp Ser Leu Thr Ala Glu Ala Met Thr Ala 50 55 60 <210> SEQ ID NO 28 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: mitrochondrial localization sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Eur. J. Biochem. <304> VOLUME: 165 <306> PAGES: 1-6 <307> DATE: 1987 <400> SEQUENCE: 28 Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Leu Ala Thr Val 1 5 10 15 Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Asn Gln Leu 20 25 30 Gln Gln Gln Gln Gln Arg Gly Lys Lys 35 40 <210> SEQ ID NO 29 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: endoplasmic reticulum sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Royal Society London Transactions B <304> VOLUME: B <306> PAGES: 1-10 <307> DATE: 1992 <400> SEQUENCE: 29 Lys Asp Glu Leu 1 <210> SEQ ID NO 30 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: adenovirus <300> PUBLICATION INFORMATION: <303> JOURNAL: EMBO J. <304> VOLUME: 9 <306> PAGES: 3153-3153 <307> DATE: 1990 <400> SEQUENCE: 30 Leu Tyr Leu Ser Arg Arg Ser Phe Ile Asp Glu Lys Lys Met Pro 1 5 10 15 <210> SEQ ID NO 31 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: farnesylation sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Nature <304> VOLUME: 302 <306> PAGES: 33-33 <307> DATE: 1983 <400> SEQUENCE: 31 Leu Asn Pro Pro Asp Glu Ser Gly Pro Gly Cys Met Ser Cys Lys Cys 1 5 10 15 Val Leu Ser <210> SEQ ID NO 32 <211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: peroxisome matrix sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Proc. Natl. Acad. Sci. U.S.A. <304> VOLUME: 4 <306> PAGES: 3264-3264 <307> DATE: 1987 <400> SEQUENCE: 32 Ser Lys Leu 1 <210> SEQ ID NO 33 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: geranylgeranylation sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Proc. Natl. Acad. Sci. U.S.A. <304> VOLUME: 91 <306> PAGES: 11963-11963 <307> DATE: 1994 <400> SEQUENCE: 33 Leu Thr Glu Pro Thr Gln Pro Thr Arg Asn Gln Cys Cys Ser Asn 1 5 10 15 <210> SEQ ID NO 34 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: destruction sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: EMBO J. <304> VOLUME: 1 <306> PAGES: 3053-3053 <307> DATE: 1996 <400> SEQUENCE: 34 Arg Thr Ala Leu Gly Asp Ile Gly Asn 1 5 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: secretory sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: J. Immunol. <304> VOLUME: 155 <306> PAGES: 3946-3946 <307> DATE: 1995 <400> SEQUENCE: 35 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser 20 <210> SEQ ID NO 36 <211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: secretory sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Nucleic Acids Res. <304> VOLUME: 7 <306> PAGES: 30-30 <307> DATE: 1979 <400> SEQUENCE: 36 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr 20 25 <210> SEQ ID NO 37 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: secretory sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Nature <304> VOLUME: 284 <306> PAGES: 26-26 <307> DATE: 1980 <400> SEQUENCE: 37 Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu 1 5 10 15 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn 20 25 <210> SEQ ID NO 38 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: secretory sequence <300> PUBLICATION INFORMATION: <303> JOURNAL: Proc. Natl. Acad. Sci. U.S.A. <304> VOLUME: 80 <306> PAGES: 3563-3563 <400> SEQUENCE: 38 Met Lys Ala Lys Leu Leu Val Leu Leu Tyr Ala Phe Val Ala Gly Asp 1 5 10 15 Gln Ile <210> SEQ ID NO 39 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: secretory signal sequence <400> SEQUENCE: 39 Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu Ala 1 5 10 15 Cys Ala Gly Asn Phe Val His Gly 20 <210> SEQ ID NO 40 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism: stability sequence <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (3)..(4) <223> OTHER INFORMATION: The amino acid at position 3 is any amino acid. <400> SEQUENCE: 40 Met Gly Xaa Gly Gly Pro Pro 1 5 <210> SEQ ID NO 41 <211> LENGTH: 2 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: linker consensus <400> SEQUENCE: 41 Gly Ser 1 <210> SEQ ID NO 42 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: linkder consensus <400> SEQUENCE: 42 Gly Ser Gly Gly Ser 1 5 <210> SEQ ID NO 43 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: linker consensus <400> SEQUENCE: 43 Gly Gly Gly Ser 

We claim:
 1. A method for forming an expression shuttle vector comprising an heterologous nucleic acid insert and capable of expressing said insert in a mammalian cell, comprising: (a) transforming yeast with a shuttle vector which shuttle vector comprises: (i) an origin of replication functional in yeast; (ii) a selectable gene functional in yeast; (iii) a promoter functional in a mammalian cell and capable of directing transcription of a polypeptide coding sequence operably linked downstream of said promoter; and (iv) an insertion site for an heterologous nucleic acid; wherein said insertion site is an homologous recombination site comprising a first nucleic acid sequence and a second nucleic acid sequence, which first and second nucleic acid sequences are contiguous, and wherein said first and second nucleic acid sequences taken separately correspond to a nucleic acid sequence at the 5′ end of said heterologous nucleic acid and a nucleic acid sequence at the 3′ end of said heterologous nucleic acid, respectively, and wherein said first and second nucleic acid sequences taken separately comprise nucleic acid sequences of from about 10 to about 100 nucleotides in length; (b) transforming yeast with a vector comprising an heterologous nucleic acid flanked by said first nucleic acid sequence and said second nucleic acid sequence; and (c) allowing said shuttle vector to recombine so as to insert said heterologous nucleic acid into said shuttle vector at said homologous recombination site.
 2. A method according to claim 1, further comprising isolating said shuttle vector from said yeast.
 3. A method for expressing an heterologous nucleic acid in a mammalian cell, comprising forming an expression shuttle vector comprising an heterologous nucleic acid using the method of claim 2 and further comprising introducing said shuttle vector to a mammalian cell for expression of said heterologous nucleic acid. 